A large number of human genetic diseases result from mutations that cause premature termination of the synthesis of proteins encoded by mutant genes. Currently, hundreds of such nonsense mutations are known, and several where shown to account for certain cases of fatal diseases, including cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), Tay-Sachs, and more. For many of those diseases there is presently no effective treatment. In the last several years, it was shown that some aminoglycoside antibiotics (including gentamicin) have the ability to allow the mammalian ribosome to selectively read past a false-stop signal, but not a normal termination signal, and generate full-length functional proteins. However, high toxicity of these drugs in humans limits their therapeutic use. The main objective of this research is to develop novel aminoglycosides that will have efficient termination suppression activity, and at the same time will have reduced toxicity against mammalian cells. To date, the clinical application of aminoglycosides is limited to their use as antibacterial drugs, and no efforts have been made to optimize their activity as stop codon read-through inducers. Toward these ends, a collaborative effort is under way between several different laboratories and combines expertise of number of complementary disciplines to synthesize and elucidate the structure-activity- toxicity relationships of the designed drugs. The hypothesis behind the proposal is to separate the elements of the aminoglycosides structures that cause toxicity from those that are required for inducing nonsense suppression: designed structures exhibiting extensive specificity and selectivity for the cytoplasmic rRNA A site can decrease the functional dosing ranges and subsequently decrease the anticipated toxicity, including deleterious effects on mitochondrial protein synthesis machinery, making them potential drugs for the treatments of human genetic disorders. Our hypothesis is based on a series of recent observations in which we have shown that by reducing the specificity to prokaryotic ribosome and as such wiping away from aminoglycosides their "natural" antibacterial activity we reduce their action on eukaryotic mitochondrial protein synthesis machinery and as such significantly reduce their toxic effects on humans. Substantial therapeutic and economic benefits are anticipated from this study. The designed structures presented here are simple for preparation and the preliminary tests already discovered some variants with lower toxicity and greater read-through efficacy to restore functional CFTR protein from the mutant gene both in vitro and in vivo, including models closely predictive of results with human CF subjects, than those of gentamicin. Fulfillment of the goals of this project will provide the necessary knowledge and tools for uncovering new structures, which may act as novel drugs. PUBLIC HEALTH RELEVANCE: Many human genetic diseases including cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome, Hurler syndrome and numerous types of cancer are caused by nonsense mutations. According to the Human Gene Mutation Database, nonsense mutations represent about 12% of all mutations reported, and for many of those diseases there is presently no effective treatment. The proposed research aims to develop potent aminoglycosides derivatives with high potential for immediate therapeutic application for treatment of genetic diseases caused by nonsense mutations.